Fluorescence is one of the most popular spectroscopic techniques currently being applied to the study of protein structure. dynamics and function. Available evidence points clearly to the dominant role of the protein matrix in determining the fluorescence properties of protein-embedded fluorophores but the mechanisms underlying the effects of the protein matrix are unclear. This proposal focuses on determining the molecular basis underlying the heterogeneous fluorescence intensity decay of tryptophan in proteins and on a detailed analysis of tryptophan side chain dynamics. The hypotheses to be tested are that short range interactions with the protein matrix are the principal determinants of the heterogeneity in pi; that water plays a major role in the temperature dependence of trp fluorescence and in apparent fluorescence-detected dipolar relaxation; that picosecond motions of the trp side chain occur on a time scale similar to those predicted from molecular dynamics simulations. To probe these issues. we will use primarily: (i) measurements of picosecond time resolved fluorescence intensity decay and multi frequency phase fluorometry to determine pi and r(t); (ii) 13C-NMR relaxation measurements on proteins labeled specifically with 13C-trp to measure "local" and global rotational correlation times (pi(e) and pi(m) respectively); (iii) molecular dynamics, minimum perturbation mapping and umbrella sampling simulations to examine the probability that specific side chain configurations exist, and their dynamics; and (iv) mathematical models to evaluate the likelihood of discrete lifetimes or distribution of pi. Experiments will be conducted mostly on proteins, each bearing a single trp residue of known tertiary structure, most of which are amenable to mutation through recombinant DNA techniques. The results should have a substantial influence on fluorescence studies of the effects of ligands (drugs) on protein structure and function, particularly proteins not amenable to detailed analysis by other spectroscopic techniques.